Translation initiation is a major target of gene regulation in eukaryotes. Internal ribosome entry sites (IRES) are RNA sequences that drive a non-canonical mechanism of translation initiation, bypassing the need for a 5'cap on the messenger RNA (mRNA). IRESs are central players in the infection cycles of many important viruses (e.g. hepatitis C, hepatitis A, poliovirus, HIV-1) and are significant regulators of gene expression. Many IRESs possess higher-order structures that interact with and manipulate the translation machinery, but our understanding of how they work is rudimentary. Thus, our knowledge of the viruses that employ IRESs remains incomplete, and our ability to exploit IRESs as drug targets is limited. Furthermore, because some IRESs are known to interact directly with the ribosome to alter its conformation and manipulate its function, IRESs offer a window into how ribosomes work, how structured RNAs can bind to ribosomes, and how ribosome function can be altered. The intergenic (IGR) IRESs of the Dicistroviridae provide a powerful model system to explore these phenomena. We propose to build upon our long-standing studies on the intergenic (IGR) IRESs of the Dicistroviridae, which drive a highly streamlined mode of ribosome recruitment and activation. Over the last few years, we have made discoveries that lead to a detailed structure-based model of IGR IRES function; we are now poised to test that model and explain the function of an IRES in unprecedented detail. We propose to do this with three specific aims. First, we will employ single molecule FRET (smFRET) to observe the motions of IRES RNAs, tRNAs, and ribosome components within an initiating complex. By watching how the structured IRES drives specific movements on the ribosome and comparing these motions to those of canonical elongation, we will gain insight at a level not yet achieved. Second, we will explore conformational changes within the IRES RNA structure itself, linking these dynamic changes to global changes in the initiating complex and in so doing, turn static pictures into dynamic pathways. Third, we will solve the structures of IRES-ribosome complexes by x-ray crystallography, which should reveal heretofore unseen intimate ribosome-IRES interactions that underlie conformational changes. These three independent but complementary aims promise to yield a cohesive, high-resolution, and dynamic mechanistic picture of how an IRES RNA manipulates a fundamental biological machine, lending wide-ranging insight into universal biological processes.